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Engineered vessels: importance of the extracellular matrix
Engineered Vessels: Importance of the Extracellular Matrix A. Solan, V. Prabhakar, and L. Niklason
V
ASCULAR DISEASE, especially that affecting the coronary arteries and other small-diameter vessels, is the leading cause of adult death in the Western world today.1 Current treatment methods for arterial failure include surgical replacement using autologous veins and arteries, as well as biocompatible synthetic materials for bypass grafts. However, a large population of patients in need of arterial bypass surgery suffer from insufficient venous or arterial bypass material. Past work has demonstrated the feasibility of growing functional autologous engineered arteries in animals.2 Inherent to the problem of developing an artificial blood vessel is the question of integrity of design. The artery must be of sufficient strength to withstand the hemodynamic pressures exerted by the body. Much of the mechanical strength of blood vessels comes from the extracellular matrix (ECM).3 Smooth muscle cells within the arterial wall lay down collagen and elastin, and deposition and subsequent remodeling of these elements help to determine many of the mechanical characteristics of blood vessels. For example, breakdown of the fibrillar collagens type I and III in the ECM is mediated, in part, by matrix metalloproteinase type I (MMP-1), which acts as a collagenase to transect the collagen fibrils.4 The ongoing interplay of collagen deposition and matrix breakdown determines the ultimate mechanical integrity of vessels over time. The growth of engineered vessels is affected by several parameters. Culture time,5 medium composition,2 and scaffold material6 all alter the properties of developing engineered arteries. One parameter that has not been studied in detail is the effect of applied pulsatile stress during culture on development of a functional artery. We investigated whether different pulse rates affect the ECM composition as it relates to collagen deposition and MMP-1 production. By examining collagen content and localization within the vessel, as well as MMP-1 level, better insight may be gained into optimal culture conditions for engineered blood vessels. MATERIALS AND METHODS Vascular smooth muscle cells were isolated from the carotid artery of Yucatan miniature swine. Cells were seeded onto tubular scaffolds that were 3 mm in diameter, 7 cm in length, and 0.2 mm in width made from polyglycolic acid (PGA) mesh.2 Scaffolds were cultured in bioreactors under various pulsatile conditions for a period of 8 weeks. Pulse rates that were examined included static conditions [0 beats per minute (bpm)], “adult” heart rate (90 bpm), 0041-1345/01/$–see front matter PII S0041-1345(00)01909-6
and “fetal” heart rate (165 bpm). Culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 10% porcine serum, basic fibroblast growth factor, platelet-derived growth factor-BB, penicillin G, HEPES, ascorbic acid, CuSO4, proline, alanine, and glycine. During culture, samples of medium were taken from the bioreactors for MMP-1 quantification assays. At the end of 8 weeks of culture, segments of the vessels were harvested and were fixed in formalin and then paraffin embedded for histological processing. Matching segments were immediately frozen for analysis of collagen content.7
RESULTS Histological Analysis
Hematoxylin and eosin (H&E) and Masson’s Trichrome stain were used on cross sections for the nonpulsed porcine vessels and vessels grown at 165 bpm (Fig 1). Masson’s stain was used to determine the presence of collagen in the preserved cross sections of the vessel wall. Collagen was found throughout the vessel walls of all engineered arteries examined. This is consistent with previous reports of collagen synthesis supported by various additives, including ascorbic acid and amino acid supplementation.2,8 MMP-1 Quantification
An ELISA-based assay (Amersham-Pharmacia, Piscataway, NJ) was used to determine the total amount of MMP-1 in bioreactor medium of each engineered vessel over the course of cultivation. We have found that this kit crossreacts with porcine enzyme isoforms. The amounts of MMP-1 present in bioreactor culture media were determined for the duration of culture. MMP-1 levels were analyzed every 2 to 3 days, and average values were calculated for the 8-week period. Results indicated that the level of MMP-1 was higher for vessels grown under pulsed conditions (Fig 2). Engineered arteries grown with an adult heart rate (90 bpm) had the highest MMP-1 levels of the three pulse rates and differed significantly from MMP-1 levels of both the vessels grown at 165 bpm (P ⬍ .0001) and from the vessels grown at 0 bpm (P ⬍ .0001). Engineered arteries grown at 165 bpm had MMP-1 levels From Duke University, Durham, North Carolina. Address reprint requests to Dr L. Niklason, Room 136 Hudson Hall, Duke University, Research Drive at Science Drive, Durham, NC 27708. E-mail: [email protected]. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
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Transplantation Proceedings, 33, 66–68 (2001)
ENGINEERED VESSELS
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Fig 1. (A) H&E stain of nonpulsed vessel. (B) Masson’s trichrome stain of nonpulsed vessel. (C) H&E stain of vessel grown at 165-bpm pulse rate. (D) Masson’s trichrome stain of vessel grown at 165-bpm pulse rate.
that did not differ significantly from the vessels grown under nonpulsed conditions (P ⬎ .05). Collagen Content
Frozen segments of the vessel were analyzed for total collagen content using a spectrophotometric method.7 The
blood vessel segments were lyophilized to obtain a dry weight and then subjected to papain digest, followed by acid hydrolysis in 6 N HCl at 115°C for 18 hours. The resulting digests were analyzed for hydroxyproline content as a reflection of total collagen in the construct by reaction with p-dimethylaminobenzaldehyde and chloramine-T. Collagen
Fig 2. MMP-1 medium levels by ELISA and collagen contents (percent of dry weight) for nonpulsed, 90-bpm, and 165-bpm vessels. 1 ⫽ nonpulsed MMP-1 (n ⫽ 22), 2 ⫽ 90-bpm MMP-1 (n ⫽ 32), 3 ⫽ 165-bpm MMP-1 (n ⫽ 29), 4 ⫽ nonpulsed collagen (n ⫽ 7), 5 ⫽ 90-bpm collagen (n ⫽ 2), 6 ⫽ 165-bpm collagen (n ⫽ 5). MMP-1 values for the nonpulsed and the 90-bpm vessels are the average of 8 weeks of medium samples. MMP-1 data for the 165-bpm vessels is an average of three different vessels. For one of the vessels, only the first 3 weeks of data were available, and for the remaining two vessels, only the final 3 weeks of data were available.
68
was determined using a ratio of hydroxyproline to collagen of 1 to 10. Results indicate that engineered porcine blood vessels grown at 90 bpm had more collagen (58.1 ⫾ 7.3% of dry weight) than vessels grown at either 165 bpm or under nonpulsed conditions. Engineered arteries grown at 165 bpm contained the least amount of collagen (39.1 ⫾ 16.9% of dry weight). However, none of the collagen content data differed significantly between the three pulse rates (P ⬎ .9). DISCUSSION
Total MMP-1 was found to be higher in culture medium of vessels undergoing pulsatile stress and was lower in vessels that were not pulsed. However, collagen content was found to be lowest in the same engineered blood vessels grown under fetal (165 bpm) pulsatile conditions. Since other investigators have demonstrated that cyclic strain can increase the activity of MMPs in engineered blood vessels,9 it is possible that the pulsatile culture conditions induce a higher rate of collagenolysis in the porcine engineered arteries. By increasing the breakdown of fibrillar collagen, active MMP-1 may contribute to the production of engineered vessels that are ultimately weaker and have less tensile strength than their native counterparts. This observation is in contrast to results observed with bovine vessels, which are more mechanically robust under pulsatile growth conditions than nonpulsed engineered arteries.2 Engineered arteries grown at 90 bpm had higher levels of MMP-1 than either those vessels grown at 165 bpm or nonpulsed vessels. However, the same vessels grown at 90 bpm contained more collagen than arteries grown under either of the other culture conditions. While these results may appear contradictory, it is important to note that in this study, only MMP-1 levels were measured. The MMP-1 assay that was utilized detects all forms of MMP-1, including those bound to tissue inhibitors of metalloproteinses (TIMPs). TIMPs are naturally occurring compounds that
SOLAN, PRABHAKAR, AND NIKLASON
are secreted by smooth muscle cells and that bind stoichiometrically to MMPs to inhibit their activity. Thus, while total MMP-1 levels were higher in the medium of blood vessels grown at 90 bpm, this may not reflect higher MMP-1 activity. Further characterization of this system, including measurement of TIMP levels and MMP-1 activity in both culture medium and engineered tissue, is required to gain better understanding of this system. In addition, the small sample size for the 90-bpm arteries (n ⫽ 2) indicates that further experimentation with a larger sample population may clarify our results. We have studied some of the effects of cyclic strain on the wall composition of porcine engineered arteries. In particular, components that affect the mechanical integrity of the blood vessel, such as collagen and MMP-1, appear to be directly affected by the pulse rate. Of course, pulse rate alone does not determine the remodeling that engineered arteries experience in culture, and much work remains to be done to better understand the balance between matrix deposition and breakdown. As this process is further studied, additional insights will be gained. REFERENCES 1. Arteriosclerosis: Report of the working group on arteriosclerosis of the National Heart, Lung, and Blood Institute. Washington DC: Department of Health and Human Services; 1981, p 2 2. Niklason LE, Gao J, Abbott WM, et al: Science 284:489, 1999 3. Cox RH: Am J Phys 234:H533, 1978 4. Moses MA, Marikovsky M, Harper JW, et al: J Cell Bio 60:379, 1996 5. Niklason LE, Abbott WA, Gao J, et al: J Vasc Surg 2000 (in press) 6. Prabhakar V: (submitted) 7. Woessner JF: Arch Biochem Biophys 93:440, 1961 8. L’Heureux N, Paquet S, Labbe R, et al: FASEB J 12:47, 1998 9. Chesler NC, Ku DK, Galis ZS: Am J Phys 277:H2002, 1999
V
ASCULAR DISEASE, especially that affecting the coronary arteries and other small-diameter vessels, is the leading cause of adult death in the Western world today.1 Current treatment methods for arterial failure include surgical replacement using autologous veins and arteries, as well as biocompatible synthetic materials for bypass grafts. However, a large population of patients in need of arterial bypass surgery suffer from insufficient venous or arterial bypass material. Past work has demonstrated the feasibility of growing functional autologous engineered arteries in animals.2 Inherent to the problem of developing an artificial blood vessel is the question of integrity of design. The artery must be of sufficient strength to withstand the hemodynamic pressures exerted by the body. Much of the mechanical strength of blood vessels comes from the extracellular matrix (ECM).3 Smooth muscle cells within the arterial wall lay down collagen and elastin, and deposition and subsequent remodeling of these elements help to determine many of the mechanical characteristics of blood vessels. For example, breakdown of the fibrillar collagens type I and III in the ECM is mediated, in part, by matrix metalloproteinase type I (MMP-1), which acts as a collagenase to transect the collagen fibrils.4 The ongoing interplay of collagen deposition and matrix breakdown determines the ultimate mechanical integrity of vessels over time. The growth of engineered vessels is affected by several parameters. Culture time,5 medium composition,2 and scaffold material6 all alter the properties of developing engineered arteries. One parameter that has not been studied in detail is the effect of applied pulsatile stress during culture on development of a functional artery. We investigated whether different pulse rates affect the ECM composition as it relates to collagen deposition and MMP-1 production. By examining collagen content and localization within the vessel, as well as MMP-1 level, better insight may be gained into optimal culture conditions for engineered blood vessels. MATERIALS AND METHODS Vascular smooth muscle cells were isolated from the carotid artery of Yucatan miniature swine. Cells were seeded onto tubular scaffolds that were 3 mm in diameter, 7 cm in length, and 0.2 mm in width made from polyglycolic acid (PGA) mesh.2 Scaffolds were cultured in bioreactors under various pulsatile conditions for a period of 8 weeks. Pulse rates that were examined included static conditions [0 beats per minute (bpm)], “adult” heart rate (90 bpm), 0041-1345/01/$–see front matter PII S0041-1345(00)01909-6
and “fetal” heart rate (165 bpm). Culture medium consisted of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 10% porcine serum, basic fibroblast growth factor, platelet-derived growth factor-BB, penicillin G, HEPES, ascorbic acid, CuSO4, proline, alanine, and glycine. During culture, samples of medium were taken from the bioreactors for MMP-1 quantification assays. At the end of 8 weeks of culture, segments of the vessels were harvested and were fixed in formalin and then paraffin embedded for histological processing. Matching segments were immediately frozen for analysis of collagen content.7
RESULTS Histological Analysis
Hematoxylin and eosin (H&E) and Masson’s Trichrome stain were used on cross sections for the nonpulsed porcine vessels and vessels grown at 165 bpm (Fig 1). Masson’s stain was used to determine the presence of collagen in the preserved cross sections of the vessel wall. Collagen was found throughout the vessel walls of all engineered arteries examined. This is consistent with previous reports of collagen synthesis supported by various additives, including ascorbic acid and amino acid supplementation.2,8 MMP-1 Quantification
An ELISA-based assay (Amersham-Pharmacia, Piscataway, NJ) was used to determine the total amount of MMP-1 in bioreactor medium of each engineered vessel over the course of cultivation. We have found that this kit crossreacts with porcine enzyme isoforms. The amounts of MMP-1 present in bioreactor culture media were determined for the duration of culture. MMP-1 levels were analyzed every 2 to 3 days, and average values were calculated for the 8-week period. Results indicated that the level of MMP-1 was higher for vessels grown under pulsed conditions (Fig 2). Engineered arteries grown with an adult heart rate (90 bpm) had the highest MMP-1 levels of the three pulse rates and differed significantly from MMP-1 levels of both the vessels grown at 165 bpm (P ⬍ .0001) and from the vessels grown at 0 bpm (P ⬍ .0001). Engineered arteries grown at 165 bpm had MMP-1 levels From Duke University, Durham, North Carolina. Address reprint requests to Dr L. Niklason, Room 136 Hudson Hall, Duke University, Research Drive at Science Drive, Durham, NC 27708. E-mail: [email protected]. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
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Transplantation Proceedings, 33, 66–68 (2001)
ENGINEERED VESSELS
67
Fig 1. (A) H&E stain of nonpulsed vessel. (B) Masson’s trichrome stain of nonpulsed vessel. (C) H&E stain of vessel grown at 165-bpm pulse rate. (D) Masson’s trichrome stain of vessel grown at 165-bpm pulse rate.
that did not differ significantly from the vessels grown under nonpulsed conditions (P ⬎ .05). Collagen Content
Frozen segments of the vessel were analyzed for total collagen content using a spectrophotometric method.7 The
blood vessel segments were lyophilized to obtain a dry weight and then subjected to papain digest, followed by acid hydrolysis in 6 N HCl at 115°C for 18 hours. The resulting digests were analyzed for hydroxyproline content as a reflection of total collagen in the construct by reaction with p-dimethylaminobenzaldehyde and chloramine-T. Collagen
Fig 2. MMP-1 medium levels by ELISA and collagen contents (percent of dry weight) for nonpulsed, 90-bpm, and 165-bpm vessels. 1 ⫽ nonpulsed MMP-1 (n ⫽ 22), 2 ⫽ 90-bpm MMP-1 (n ⫽ 32), 3 ⫽ 165-bpm MMP-1 (n ⫽ 29), 4 ⫽ nonpulsed collagen (n ⫽ 7), 5 ⫽ 90-bpm collagen (n ⫽ 2), 6 ⫽ 165-bpm collagen (n ⫽ 5). MMP-1 values for the nonpulsed and the 90-bpm vessels are the average of 8 weeks of medium samples. MMP-1 data for the 165-bpm vessels is an average of three different vessels. For one of the vessels, only the first 3 weeks of data were available, and for the remaining two vessels, only the final 3 weeks of data were available.
68
was determined using a ratio of hydroxyproline to collagen of 1 to 10. Results indicate that engineered porcine blood vessels grown at 90 bpm had more collagen (58.1 ⫾ 7.3% of dry weight) than vessels grown at either 165 bpm or under nonpulsed conditions. Engineered arteries grown at 165 bpm contained the least amount of collagen (39.1 ⫾ 16.9% of dry weight). However, none of the collagen content data differed significantly between the three pulse rates (P ⬎ .9). DISCUSSION
Total MMP-1 was found to be higher in culture medium of vessels undergoing pulsatile stress and was lower in vessels that were not pulsed. However, collagen content was found to be lowest in the same engineered blood vessels grown under fetal (165 bpm) pulsatile conditions. Since other investigators have demonstrated that cyclic strain can increase the activity of MMPs in engineered blood vessels,9 it is possible that the pulsatile culture conditions induce a higher rate of collagenolysis in the porcine engineered arteries. By increasing the breakdown of fibrillar collagen, active MMP-1 may contribute to the production of engineered vessels that are ultimately weaker and have less tensile strength than their native counterparts. This observation is in contrast to results observed with bovine vessels, which are more mechanically robust under pulsatile growth conditions than nonpulsed engineered arteries.2 Engineered arteries grown at 90 bpm had higher levels of MMP-1 than either those vessels grown at 165 bpm or nonpulsed vessels. However, the same vessels grown at 90 bpm contained more collagen than arteries grown under either of the other culture conditions. While these results may appear contradictory, it is important to note that in this study, only MMP-1 levels were measured. The MMP-1 assay that was utilized detects all forms of MMP-1, including those bound to tissue inhibitors of metalloproteinses (TIMPs). TIMPs are naturally occurring compounds that
SOLAN, PRABHAKAR, AND NIKLASON
are secreted by smooth muscle cells and that bind stoichiometrically to MMPs to inhibit their activity. Thus, while total MMP-1 levels were higher in the medium of blood vessels grown at 90 bpm, this may not reflect higher MMP-1 activity. Further characterization of this system, including measurement of TIMP levels and MMP-1 activity in both culture medium and engineered tissue, is required to gain better understanding of this system. In addition, the small sample size for the 90-bpm arteries (n ⫽ 2) indicates that further experimentation with a larger sample population may clarify our results. We have studied some of the effects of cyclic strain on the wall composition of porcine engineered arteries. In particular, components that affect the mechanical integrity of the blood vessel, such as collagen and MMP-1, appear to be directly affected by the pulse rate. Of course, pulse rate alone does not determine the remodeling that engineered arteries experience in culture, and much work remains to be done to better understand the balance between matrix deposition and breakdown. As this process is further studied, additional insights will be gained. REFERENCES 1. Arteriosclerosis: Report of the working group on arteriosclerosis of the National Heart, Lung, and Blood Institute. Washington DC: Department of Health and Human Services; 1981, p 2 2. Niklason LE, Gao J, Abbott WM, et al: Science 284:489, 1999 3. Cox RH: Am J Phys 234:H533, 1978 4. Moses MA, Marikovsky M, Harper JW, et al: J Cell Bio 60:379, 1996 5. Niklason LE, Abbott WA, Gao J, et al: J Vasc Surg 2000 (in press) 6. Prabhakar V: (submitted) 7. Woessner JF: Arch Biochem Biophys 93:440, 1961 8. L’Heureux N, Paquet S, Labbe R, et al: FASEB J 12:47, 1998 9. Chesler NC, Ku DK, Galis ZS: Am J Phys 277:H2002, 1999